New use of an extract of plant origin of globularia and method for obtaining said extract by in vitro plant culture

ABSTRACT

It can be used as an active agent in a composition for preventing and/or treating skin aging by stimulating detoxification reactions and cellular regeneration thanks to a hormetic type response. With such a composition, improvement of the visual appearance of the skin, radiance and transparency of complexion is obtained for example.

The present invention concerns a novel extract of plant origin, acomposition containing it, a preferred method for obtaining this extractby in vitro plant culture and topical uses in the domain of cosmeticsand personal hygiene and care products.

The present invention concerns more generally the cosmetics,pharmaceutical, and cosmeceutical industries, that produce and/or useextracts of plant origin for preventing of treating disorders andpathologies of skin, scalp, mucous and appendages (such as hair,eyelashes, eyebrows, nails or hairs) of animal or human mammals.

In cosmetics, there are many ways to improve the overall condition ofthe skin, hair and nails, including imparting or restoring radiance,hydration, pigmentation or depigmentation, protection against externalaggressions such as UV radiations or coldness, calming irritation,redness, acne, reducing micro-oedemas (such as bags under the eyes),reducing dark circles, signs of aging such as wrinkles, fine lines,pigmentation, restoring suppleness and elasticity, treating hair loss,acting on the adipose tissues, adding volume, density, improving thetexture, etc.

Extract of plant origin means according to the invention, an extractthat can be obtained directly from a plant or that can be obtained byplant culture.

Since a long time and in all parts of the world, the plant kingdom is anabundant and widely used source of biologically active substances, inparticular in the pharmaceutical and cosmetics fields. Possibilities areinfinite.

The aim of the present invention is to provide a new extract of plantorigin for the cosmetic and dermopharmacy industry always in demand ofnew active ingredients.

To this aim, the present invention proposes an extract of plant origincharacterized in that it is obtained from a plant of Globularia genus.

The skin is subject to many external stresses: UV, electromagneticwaves, oxidants, pollutants, biological agents. These stresses induce,among other deleterious effects, micro-inflammatory phenomena that aremore or less chronic and the formation of toxic products in epidermalcells or at their periphery. Among these products are thepre-inflammatory mediators and some growth factors that cause a localover-densification of epidermal nerve endings associated with ahyper-reactivity to external stimuli. This results in lowering thethreshold of the skin regarding these stimuli. It becomes verysensitive, generating discomfort but also most of the time transientrednesses.

In addition, the skin gradually accumulates debris of various origins,composed of altered proteins, carbohydrates and lipids. These components(carbonylated proteins, lipoperoxides, lipofuscin, . . . ) will also actas toxins that disrupt the physiology and cellular homeostasis andmodify fragile metabolic balances. Over time, they alter mitochondrialrespiration, membrane integrity, enzymatic activity, and detoxificationmechanisms such as the proteasome. Such changes can cause changes inbehavior of certain cells such as stem cells. As a matter of fact, thelatter are in a particular environment in which the change may lead tothe weakening of their character and their progenitor phenotype (cellsize larger, reduction of the multiplier capacity etc.), making themless able to hold their role in the renewal of keratinocytes.

Experimental data show that mild stress received in an early stage candelay degradation and therefore skin aging, helping the body to laterdeal with more severe stress (external events). This phenomenon iscalled hormesis. It is based on the activation of detoxificationmechanisms and cellular defense in response to a mild stress or inresponse to substances which are said to have a hormetic effect(molecules called hormetins). The hormesis will contribute to a betteradaptation of the cell and thus of the body with regards to subsequentstresses, allowing it to fight more effectively against the harmfuleffects mentioned above.

It is thus very interesting from a cosmetic, dermo-pharmaceutical andcosmeceutical point of view to mimic the effects of hormesis to improvethe physiology of the skin and allow it to better fight against futurestresses and thus against skin ageing. For a better understanding ofthis hormetic effect, it can be spoken of an anti-ageing vaccine.However, the hormetic effect, although showing its effect on long term(anti-aging), will also improve rapidly the condition of the skin (onthe short term) by giving it a lower sensitivity to external stressesand by providing more transparency and brightness.

It is this set of original and unexpected properties that has beenparticularly shown for the extract according to the invention through aseries of tests that are developed below.

Thus, the detailed description given below, shows that this extract hasin vivo properties completely unexpected and original in the targetedtechnical fields, in particular an improvement of skin transparency andcutaneous reactivity, producing a remarkable effect on the visualquality of the skin: radiance, clarity, purity and homogeneity ofcomplexion, smoothness aspect, etc. thanks in particular to astimulation of the hormetic defenses of the skin.

Globularia genus belongs to the Globulariaceae family, and comprisesseveral species, including those known: Globularia alypum, Globulariabisnagarica, Globularia cordifolia, Globularia nudicaulis, Globulariavulgaris, Globularia gracilis, Globularia repens and Globulariavalentina. The Applicant has been particularly interested in the speciesGlobularia cordifolia.

The present invention therefore provides an extract of plant origin ofthe Globularia plant, preferably Globularia cordifolia, for use as acosmetic active ingredient to improve the general condition of the skinand appendages, especially more particularly for use as an ingredientcosmetic active agent for preventing and/or treating cutaneous aging bystimulating detoxification reactions and cellular regeneration thanks toan hormetic type response. Examples are given below showing that theGlobularia extract according to the invention is effective for use as anactive ingredient, in particular for improving the radiance and/or thetransparency of complexion, for preventing and/or treating sensitiveand/or reactive skins, to prevent and/or treat redness, to preventglycation of proteins.

The extract according to the invention can be used pure or diluted in aphysiologically acceptable excipient or matrix.

Other uses are possible, whitening, moisturizing, anti-wrinkles,firming, slimming, for increasing epidermis or dermis volume, anti-acne,anti-inflammatory, etc.

Particularly in cosmetics, applications can be offered for example inthe ranges of moisturizers, cleansers, cleansing, anti-aging,antioxidant, protective, restorative (hands, feet, lips), contours(face, eyes, neck, lips), makeup skin care and for appendages, includingeyelashes, lip products, solar products, remodeling, plumping,reshaping, lipofiling (e.g. on the hands, chest, breasts), hairproducts, etc.

According to other features, the extract of the invention is preferablyobtained by in vitro plant culture, more particularly by plant cellculture or by tissue culture from cellular or tissular lines fromdifferent organs of the plant.

The invention covers also the extracts obtained from the whole plant orspecific parts of this plant.

The obtaining of extracts of plant origin by in vitro culture presentsnumerous advantages over the agro-industrial path (plant culture in openfields and subsequent extraction in factories). The obtained extractsare free of toxic substances (herbicides, pesticides, fertilizers, heavymetals and other contaminants, such as those that may come from plantparasites). Moreover, the strict control of in vitro culture conditionsreduces the risk of spontaneous variation of the strain profile andensures a reproducible secondary metabolites profile that corresponds tothe molecules of interest sought, contrary to the culture in an openfield where there is the problem of variability due to weather andgeography. Furthermore, this technology overcomes obstacles such as thenatural biological cycle of the plant and the seasonality of productionof the secondary metabolites, thus allowing a better safety and supplyrapidity. In addition, the environmental impact is minimal avoidingconsumption of arable lands and soils pollution. Furthermore,biodiversity is preserved since it takes only one plant or even a seedto initiate an in vitro culture. Finally, this technology offers theability to direct cellular metabolism toward the production of moleculesof interest (elicitation of the cultures) and to achieve some controlledand relatively rapid protocols in order to maximize yields.

Among the today existing technics in the field of in vitro plantculture, the followings can be used according to the invention:

Culture of undifferentiated cells: this method involves first thecreation of highly proliferative cell lines in agar medium. These linesare then grown in liquid medium in order to substantially increase thebiomass. At the end of the growth cycle and environmental conditions tobe defined and optimized (searching for the appropriate elicitationmedium), the cell biomass will synthesize the molecules of interest. Theculture is then stopped and extracted at the optimum time to obtain anextract, said to be of plant origin, containing a maximum amount ofmolecules of interest. Existing cell lines already available can also beused initially.

Culture of tissue or organ: this type of culture can concern the rootpart (“root culture”) or the aerial part (“shoot culture”) or thesomatic embryo (□somtic embryo□). In this type of method, there arecultures that have been transformed by genomics bacteria Agrobacteriumrhizogenes (root) or Agrobacterium tumefaciens (stems). Roots or aerialparts cultures thus transformed have a high growth rate and aregenetically very stable. They are used to synthesize the molecules ofinterest after optimizing the elicitation parameters. These moleculesare then extracted by conventional means.

In vitro micropropagation through somatic embryogenesis or vegetativemultiplication. The first step for these two techniques consists inselecting a parent plant with optimal characteristics (in terms ofgrowth and production of metabolites of interest). In embryogenesis, thenext step is the induction of callus from explants from the parentplant. These undifferentiated cells can give rise to numerous somaticembryos that it will be possible to multiply and extend throughsuccessive cycles of culture. From the most proliferative and productivelines, seedlings are regenerated in a suitable medium and extracted inat the appropriate time for optimal production of metabolites ofinterest. For vegetative multiplication, the second step consists in theinduction of stems with leaves from explants of the parent plant on asuitable culture medium thanks to its hormonal composition. Anamplification phase of the leafy stems is followed by a rooting phase toinduce roots. It is then possible to regenerate plantlets in largequantities, to grow them in a suitable medium and extract the moleculesof interest.

The present invention preferentially provides a method of manufacturingaccording to the first technique. More particularly, it provides amethod of obtaining of an extract of plant origin by in vitro plantculture, comprising from a line of undifferentiated plant cells:

-   -   A step of pre-culture, for increasing the biomass,    -   A step of culture in a bioreactor comprising a proliferation        phase and then an elicitation phase, and    -   A recovering step of said extract.

The method according to the invention is characterized in that the cellline is a line of the plant of the genus Globularia and preferably ofthe species Globularia cordifolia.

According to other features:

-   The step of pre-culture comprises the completion of pre-cultures of    increasing sizes, each pre-culture of smaller size for generating    cellular biomass in sufficient quantities to inoculate the    pre-culture of a larger size.-   The subsequent step of culture in bioreactor is achieved in a    bioreactor inoculated with the biomass produced in the pre-culture    step. After an initial phase of cell proliferation in a base medium,    a medium of elicitation of secondary metabolites is added to the    bioreactor. Culture is stopped when the desired level of secondary    metabolites has been achieved. Preferably, to achieve this level,    according to the invention, an elicitation medium deficient in    sucrose and macronutrients is used.-   The recovery step includes an extraction stage of this biomass by    any physiologically acceptable solvent, or any mixture of these    solvents. The extraction can be done by different known methods that    can be combined: heat, maceration, decoction, infusion, pressure,    leaching, ultrasonic, microwave, by lysing the cells by any chemical    or physical appropriate method. Phase separation can be achieved by    filtration or centrifugation. Alternatively, it is possible to    extract the biomass with a supercritical or subcritical fluid.    Alternatively also, it is possible to achieve a purification using    an adsorbent resin, chromatographic methods or liquid-liquid    partition.    -   According to a preferred embodiment, the cells are lysed in        order to transfer the cell contents in water. More preferably        according to the present invention, the lysis is performed by        lowering to an acidic pH. After several filtrations, a sterile        extract is obtained. Its pH is then adjusted advantageously        according to the further use of the extract, for example at pH 4        for a cosmetic application as described below.

According to the invention, the cell line may be an existing line or aline created in a preliminary stage, with said step being performedseparately in time.

According to the invention, the step of creating the cell line comprisesthe steps 1) induction of callus (clusters of undifferentiated cells),2) selection of the best callus line and 3) optimization on the selectedcallus in liquid medium of the growth parameters and production ofmolecules of interest (e.g. by elicitation).

More particularly:

-   1) The induction of callus can be done with all parts of the plant,    including leaf, fruit, root, bud, seed, stem, branch, and    meristematic tissues, and cambium; preferably according to the    invention, the undifferentiated cell culture is achieved from the    leaves of the plant.-   2) The selection of the best lines, before a transfer in liquid    medium, is performed according to the invention including in    particular to the following criteria: high proliferative capacity,    tender and friable texture, uniform color, and good dispersion in    liquid medium and long-term stability of these parameters.-   3) Optimization of secondary metabolites growth and production    characteristics in liquid medium for the selected lines through the    choice of the most appropriate media, in a first step to ensure    rapid growth of the biomass and in a second time to allow maximum    efficiency of secondary metabolites synthesis by cells at the end of    exponential phase (stop of multiplication). This second phase    corresponds to the culture elicitation and can be achieved in    different ways among those available to the skilled person. These    include: the addition to the cultivation of microbial fractions,    stress molecules known to direct cells to their secondary    metabolism, the application to the culture of a change in    temperature or pH, or an osmotic stress, the use of an    impoverishment of the environment, adding to the culture of    adsorbent resins, which in addition to eliciting the compounds of    interest can trap them. The preferred method of elicitation    according to the invention is to impoverish the culture medium in    macro-elements and sugar.

Thus, according to the invention, an original cell line is obtainedmaking it possible to produce an extract of plant origin according tothe invention, which is original and active in the desired domains, aswill be shown by the efficacy tests given below, preferably a cell lineof Globularia cordifolia.

When starting from a cell line according to the invention alreadyexisting and preserved, this cell line will be in a conventional step,prior to the pre-culture step, of induction of the undifferentiatedplant cells, initially in agar medium, then in liquid medium.

The present invention also covers an extract of plant origin obtainedconventionally by solvent extraction of the whole plant or specificparts of the plant, by supercritical fluid, micro-wave or ultrasounds,but comparative results showed superiority of the extract obtained froma cell line compared to a conventional solvent extract. These resultsare given below in the detailed description.

Another object of the present invention is a topical composition, inparticular a cosmetic, pharmaceutical or cosmeceutical composition,characterized in that it contains, in a physiologically acceptablemedium, as active agent, an effective amount of an extract of plantorigin of a plant of the genus Globularia, and more particularly of thespecies Globularia cordifolia.

According to other advantageous features of the present invention, theextract of Globularia can be combined with at least one additionalactive ingredient, in order to preferably provide an end product with awider range of properties. Additional active ingredients can be forexample selected from whitening agents, anti-redness, UV sunscreens,moisturizing, humectant, exfoliating anti-aging, anti-wrinkles and finelines, sliming, volumizing, improving the elastic properties, anti-acne,anti-inflammatory, anti-oxidant, anti-radical, propigmenting ordepigmenting, depilatories, anti-growth or promoting hair growth,peptides, vitamins agents etc. These active ingredients can be obtainedfrom plant materials such as plant extracts or products of plant cultureor fermentation. More specifically, in a cosmetic composition, theextract of plant origin according to the invention can be combined withat least one of the compounds selected from the compounds of theB3vitamin compounds such as niacinamide and tocopherol, retinoidcompounds such as retinol, hexamidine, α-lipoic acid, resveratrol orDHEA, peptides, including N-acetyl-Tyr-Arg-O-hexadecyl, Pal-VGVAPG,Pal-KTTKS, Pal-GHK, Pal-KMO2K and Pal-GQPR, which are conventionalactive ingredients used in topical cosmetic or dermo-pharmaceuticalcompositions. The present invention will be better understood in lightof the following description. The following examples illustrate theinvention without limiting it only to those applications.

Composition Preparation

The expression “physiologically acceptable medium” means according tothe present invention, without limitation, an aqueous or hydro-alcoholicsolution, a water-in-oil emulsion, an oil-in-water emulsion, amicro-emulsion, an aqueous gel, an anhydrous gel, a serum, a dispersionof vesicles, a powder.

“Physiologically acceptable” means that the compositions are suitablefor topical use in contact with mucous membranes, nails, scalp, hairs,hair and skin of mammals, particularly human, without risk of toxicity,incompatibility, instability, allergic response, and others.

“The physiologically acceptable medium” forms what is commonly calledthe excipient of the composition.

The effective amount of the extract of plant origin for implementing thepresent invention, that is to say its dosage, depends on the destinationof the topical cosmetic, pharmaceutical, cosmeceutical composition.

The effective cosmetic, pharmaceutical, cosmeceutical amount accordingto the invention, to be administered to treat a disorder or conditionand its dosage depend on various factors such as age, the state of thepatient, the severity of the disorder or condition and the mode ofadministration. An effective amount means a not toxic amount enough toachieve the desired effect.

In a cosmetic composition according to the present invention, theextract of Globularia to be present in an effective amount, is generallyin an amount ranging from 0.000001% to 15% with regard to the totalweight of the composition, more preferably between 0.0001% and 10%,depending on the destination of the composition and the desired effectmore or less pronounced.

All percentages and ratios used herein are by weight of the totalcomposition and all measurements are made at 25° C. unless it isotherwise specified.

The choice of excipient in the composition is made according to theconstraints of the active extract of Globularia (stability, solubility,etc.), and if necessary according to the dosage form intended furtherfor the composition.

The Globularia extract of the invention may be incorporated into thecomposition by means of an aqueous solution, or be dissolved by theusual physiologically acceptable solubilizers, for example and withoutlimiting to this list: ethanol, propanol, isopropanol, propylene glycol,glycerin, butylene glycol, or polyethylene glycol or any combinationthereof. It may also be interesting to solubilize the extract withemulsifiers. A powder medium can also be used.

The compositions for the present invention are generally prepared byconventional methods well known to one skilled in the art for makingtopical and oral compositions and injection compositions. Such methodsmay involve a mixture of ingredients in one or more steps to obtain auniform state, with or without heating, cooling, etc.

The different galenic forms that may contain the Globularia extract ofthe invention are all forms i.e. creams, lotions, milks or creamsointments, gels, emulsions, dispersions, solutions, suspensions,cleansers, foundations, anhydrous preparations (sticks in particular lipbalm, body and bath oils), shower and bath gels, shampoo and hair carelotions, milks or creams for skin care or hair, cleansing lotions ormilks, sunscreen lotions, milks or creams, artificial tanning lotions,milks or creams, pre shave, shaving or aftershave creams, foams, gels orlotions, makeup, lipstick, mascara or nail polish, skin □essences□serums, adhesive or absorbent materials, transdermal patches, oremollient powders, lotions, milks or creams, sprays, body and bath oils,foundation basis, ointment, emulsion, colloid, compact suspension orsolid, pencil, sprayable formulation, brushable, blush, red, eyeliner,lipliner, lip gloss, face or body powder, styling gels or mousses, nailconditioning, lip balms, skin conditioners, moisturizers, lacquers,soaps, exfoliants, astringents, depilatories agents, permanent wavingsolutions, antidandruff formulations, antiperspirant or antiperspirantcompositions, including sticks, “roll-on□ deodorants, air fresheners,sprays for the nose and etc.These compositions may also be in the formof lipsticks intended either to color the lips or to prevent them fromchapping, or makeup for eyes, eyes-shadows and foundations for the face.The compositions for the invention can include cosmetics, personal careproducts and pharmaceutical preparations. A composition in the form offoam or in the form of aerosol compositions also comprising apressurized propellant can be considered.

The compositions according to the invention may also be for oro-dentaluse, for example a toothpaste. In this case, the compositions maycontain usual adjuvants and additives for oral compositions and inparticular surfactants, thickening agents, humectants, polishing agentssuch as silica, various active ingredients such as fluorides, inparticular particularly sodium fluoride, and optionally sweeteners assodium saccharinate.

The extract according to the present invention may be in the form ofsolution, dispersion, emulsion, paste, or powder, individually or as apremix or vehicled individually or as a premix in vectors such asmacrocapsules, microcapsules, or nanocapsules, macrospheres,microspheres or nanospheres, liposomes, oleosomes or chylomicrons,macroparticles, microparticles, or nanoparticles, macrosponges,microsponges or nanosponges, microemulsions or nanoemulsions, oradsorbed on organic polymer powders, talcs, bentonites, spores or exinesand other inorganic or organic supports. The Globularia extractaccording to the present invention may be used in any form, in a formbound to or incorporated in or absorbed in or adsorbed on macro-,micro-, and nanoparticles, or macro-, micro-, and nano-capsules, for thetreatment of textiles, natural or synthetic fibers, wools, and anymaterials that may be used for clothing or underwear for day or nightintended to come into contact with the skin, handkerchiefs or cloths, toexert their cosmetic effect via this skin/textile contact and to permitcontinuous topical delivery.

Additional Ingredients

The CTFA International cosmetic ingredient dictionary & handbook (13thEd. 2010) (published by the Cosmetic, Toiletry, and FragranceAssociation, Inc., Washington, D.C.) describes a non-limited widevariety of cosmetic and pharmaceutical ingredients conventionally usedin the skin care industry that can be used as additionalingredients/compounds in the compositions for the present invention.Examples of these ingredient classes include, but are not limited to:healing agents, anti-aging agents, anti-wrinkle agents, anti-atrophyagents, skin moisturizing agents, skin smoothing agents, antibacterialagents, anti-parasitic agents, antifungal agents, fungicidal agents,fungi static agents, bactericidal agents, bacteriostatic agents,antimicrobial agents, anti-inflammatory agents, anti-pruriginous agents,anesthetic agents, antiviral agents, keratolytic agents, free radicalsscavengers, anti-seborrhea agents, antidandruff agents, the agentsmodulating differentiation, proliferation or pigmentation of the skinand agents accelerating penetration, desquamating agents, melaninsynthesis stimulating or inhibiting agents, whitening, depigmenting orlightening agents, pro-pigmenting agents, self-tanning agents,NO-synthase inhibiting agents, antioxidants, free radical scavengersand/or agents against atmospheric pollution, reactive carbonyl speciesscavengers, anti-glycation agents, tightening agents, agents stimulatingthe synthesis of dermal or epidermal macromolecules and/or capable ofinhibiting or preventing their degradation, such as for example collagensynthesis stimulating agents, elastin synthesis stimulating agents,decorin synthesis stimulating agents, laminin synthesis stimulatingagents, defensin synthesis stimulating agents, chaperone synthesisstimulating agents, aquaporin synthesis stimulation agents, hyaluronicacid synthesis stimulating agents, fibronectin synthesis stimulatingagents, sirtuin synthesis-stimulating agents, agents stimulating thesynthesis of lipids and components of the stratum corneum (ceramides,fatty acids, etc.), collagen degradation inhibiting agents elastindegradation inhibiting agents, agents stimulating fibroblastproliferation, agents stimulating keratinocyte proliferation, adipocyteproliferation stimulating agents, melanocyte proliferation stimulatingagents, keratinocyte differentiation stimulating agents, adipocytedifferentiation stimulating agents, acetylcholinesterase inhibitingagents, glycosaminoglycan synthesis stimulating agents, DNA repairagents, DNA protecting agents, anti-itching agents, agents for thetreatment and/or care of sensitive skin, firming agents, anti-stretchmark agents, astringent agents, sebum production regulating agents,dermo-relaxing agents, healing adjuvant agents, re-epithelializationstimulating agents, re-epithelialization co-adjuvant agents, cytokinegrowth factors, calming agents, anti-inflammatory agents, agents actingon capillary circulation and/or microcirculation, angiogenesisstimulating agents, vascular permeability inhibiting agents, agentsacting on cell metabolism, agents able to improve dermal-epidermaljunction, agents inducing hair growth, hair growth inhibiting orretardant agents, muscle relaxants agents, antipollution and/oranti-free radical agents, lipolysis stimulating agents, slimming agents,anti-cellulite agents, agents acting on the microcirculation, agentsacting on the energy metabolism of the cells, cleaning agents, hairconditioning agents, hair styling agents, hair growth promoters,sunscreen agents, total sunscreen agents, make-up agents, detergents,pharmaceutical products, emulsifiers, emollients, organic solvents,antiseptic agents, deodorant actives, physiologically acceptablecarriers, surfactants, abrasives, absorbents, aesthetic components suchas fragrances, pigments, dyes, colorants, natural colorants, essentialoils, touch agents, cosmetic astringents, anti-acne agents,anti-coagulation agents, anti-foaming agents, antioxidants, binders,biological additives, enzymes, enzymatic inhibitors, enzyme-inducingagents, coenzymes, chelating agents, plant extracts, plant derivatives,essential oils, marine extracts, agents obtained from a bio-fermentationor a biotechnological process, mineral salts, cell extracts, sunscreens(organic or mineral photoprotective agents active against ultraviolet Aand/or B rays), ceramides, peptides, buffers, volumizing agents,chelating agents, chemical additives, colorants, cosmetic biocides,denaturants, medical astringents, external analgesics, film formers,such as polymers, for exacerbing film-forming properties andsubstantivity of the composition, quaternary derivatives, substantivityincreasing agents, opacifying agents, pH adjusters and regulators (e.g.triethanolamine), propellants, reducing agents, sequestrants, decoloringand/or lightening agents, skin-conditioning agents (e.g., humectants,including miscellaneous and occlusive), moisture retaining agents,alphahydroxyacids, betahydroxyacids, moisturizers, epidermal hydrolyticenzymes, healing and/or calming agents, skin treating agents,anti-wrinkle agents, agents that reduce or treat bags under the eyes,exfoliating agents, thickeners, softening agents, gellifying polymers,vitamins and their derivatives, wetting agents, peeling agents, soothingagents, curative agents of the skin, lignans, preservatives (i.e.phenoxyethanol and parabens), anti UV, cytotoxic agents,anti-neoplastics, viscosity modifiers, non-volatile solvents, pearlingagents, anti-perspirant agents, depilatories, vaccine, perfumed water,skin restructuring agent (i.e. Siegesbeckia orientalis extract),excipients, charges, minerals, anti-mycobacterial agents,anti-allergenic agents, H1 or H2 antihistaminics, anti-irritants, agentsstimulating the immune system, agents inhibiting the immune system,insect repellents, lubricants, pigments or dyes, hypopigmentationagents, preservatives, light stabilizers, and mixtures thereof, as longas they are physically and chemically compatible with the otheringredients of the composition and especially with the actives of thepresent invention. Also the nature of these additional ingredientsshould not unacceptably alter the benefits of the active ingredients ofthe invention. These additional ingredients can be synthetic or naturalsuch as plants extracts, or come from a bio-fermentation process.Additional examples can be found in the CTFA Cosmetic IngredientHandbook.

Such additional active ingredient/compound can be selected from thegroup consisting of: sugar amines, glucosamine, D-glucosamine, N-acetylglucosamine, N-acetyl-D-glucosamine, mannosamine, N-acetyl mannosamine,galactosamine, N-acetyl galactosamine, B3 vitamin and its derivatives,niacinamide, sodium dehydroacetate, dehydroacetic acid and its salts,phytosterols, salicylic acid compounds, hexamidines, dialkanoylhydroxyproline compounds, soy extracts and derivatives, equol,isoflavones, flavonoids, phytantriol, farnesol, geraniol, bisabolol,peptides and their derivatives, di-, tri-, tetra-, penta-, andhexapeptides and their derivatives, lys-thr-thr-lys-ser,palmitoyl-lys-thr-thr-lys-ser, carnosine, N-acyl amino acid compounds,retinoids, retinyl propionate, retinol, retinyl palmitate, retinylacetate, retinal, retinoic acid, water-soluble vitamins, ascorbates, Cvitamin, ascorbyl glucoside, ascorbyl palmitate, magnesium ascorbylphosphate, sodium ascorbyl phosphate, vitamin B and their derivatives,B1 vitamin, B2 vitamin, B6 vitamin, B12 vitamin, K vitamin andderivatives, pantothenic acid and its derivatives, pantothenyl ethylether, panthenol and derivatives, dexpanthenol, biotin, amino acids andtheir salts and derivatives, water soluble amino acids, asparagine,alanine, indole, glutamic acid, water insoluble vitamins, A vitamin, Evitamin, vitamin F, D vitamin and mono-,di-, and tri-terpenoidscompounds, beta-ionol, cedrol, and their derivatives, water insolubleamino acids, tyrosine, tryptamine, particulate materials, butylatedhydroxytoluene, butylated hydroxyanisole, allantoin, tocopherolnicotinate, tocopherol, tocopherol esters, pal-GHK, phytosterol, hydroxyacids, glycolic acid, lactic acid, lactobionic acid, keto acids, pyruvicacid, phytic acid, lysophosphatidic acid, stilbenes, cinnamates,resveratrol, kinetin, zeatin, dimethylaminoethanol, natural peptides,soy peptides, salts of sugar acids, Mn gluconate, Zn gluconate,piroctone olamine, 3,4,4

trichlorocarbanilide, triclocarban, Zn pyrithione, hydroquinone, kojicacid, ascorbic acid, magnesium ascorbyl phosphate, ascorbyl glucoside,pyridoxine, aloe vera, terpene alcohols, allantoin, bisabolol,dipotassium glycyrrhizinate, glycerol acid, sorbitol acid,pentaerythritol acid, pyrrolidone acid and salts, dihydroxyacetone,erythrulose, glyceraldehyde, tartaraldehyde, clove oil, menthol,camphor, eucalyptus oil, eugenol, menthyl lactate, witch hazeldistillate, eicosene and vinyl pyrrolidone copolymer, iodopropylbutylcarbamate, a polysaccharide, an essential fatty acid, a salicylate,glycyrrhetinic acid, carotenoids, ceramides and pseudo-ceramides, alipid complex, oils in general of natural origin such shea butter,apricot oil, onagre oil, prune oil, palm oil, monoi oil, hydroquinone,HEPES, procysteine, O-octanoyl-6-D-maltose, the disodium salt ofmethylglycinediacetic acid, steroids such as diosgenin and derivativesof DHEA, DHEA or dehydroepiandrosterone and/or a precursor or chemicalor biological derivative thereof, N-ethyloxycarbonyl-4-para-aminophenol,blueberries extracts, phytohormones, extracts of the yeast Saccharomycescerevisiae, extracts of algae, extracts of soyabean, lupin, maize and/orpeas, alverine and its salts, in particular alverine citrate, extract ofbutcher

broom and of horse chestnut, and mixtures thereof, a metalloproteinaseinhibitor.

Further skin care and hair care active ingredients that are particularlyuseful in combination with the composition can be found in Sedermacommercial literature and on the website www.sederma.fr.

The following commercial actives can also be mentioned, as examples:betain, glycerol, Actimoist Bio 2□ (Active organics), AquaCacteen□(Mibelle AG Cosmetics), Aquaphyline□ (Silab), AquaregulK□ (Solabia),Carciline□ (Greentech), Codiavelane□ (Biotech Marine), Dermaflu□ (ArchChemicals, Inc), Hydra

low□ (Sochibo), Hydromoist L□ (Symrise), RenovHyal□ (Soliance), Seamoss□(Biotech Marine), Essenskin□ (Sederma), Moist 24□ (Sederma), Argireline□(trade name of the acetyl hexapeptide-3 of Lipotec), spilanthol or anextract of Acmella oleracea known under the name Gatuline Expression□,an extract of Boswellia serrata known under the name Boswellin□,Deepaline PVB□ (Seppic), Syn-AK□ (Pentapharm), Ameliox□, Bioxilift□(Silab), Juvinity□ (Sederma), Revidrat□ (Sederma), or mixtures thereof.

Among other plant extracts which can be combined with the extract of theinvention, there may more particularly be mentioned extracts of Ivy, inparticular English Ivy (Hedera Helix), of Bupleurum chinensis, ofBupleurum Falcatum, of arnica (Arnica Montana L), of rosemary(Rosmarinus officinalis N), of marigold (Calendula officinalis), of sage(Salvia officinalis L), of ginseng (Panax ginseng), of ginko biloba, ofSt.-John

-Wort (Hyperycum Perforatum), of butcher

-broom (Ruscus aculeatus L), of European meadowsweet (Filipendulaulmaria L), of big-flowered Jarva tea (Orthosiphon Stamincus Benth), ofalgae (Fucus Vesiculosus), of birch (Betula alba), of green tea, of colanuts (Cola Nipida), of horse-chestnut, of bamboo, of Centella asiatica,of heather, of fucus, of willow, of mouse-ear, of escine, of cangzhu, ofchrysanthellum indicum, of the plants of the Armeniacea genus,Atractylodis Platicodon, Sinnomenum, Pharbitidis, Flemingia, of Coleussuch as C. Forskohlii, C. blumei, C. esquirolii, C. scutellaroides, C.xanthantus and C. Barbatus, such as the extract of root of Coleusbarbatus, extracts of Ballote, of Guioa, of Davallia, of Terminalia, ofBarringtonia, of Trema, of antirobia, cecropia, argania, dioscoreae suchas Dioscorea opposita or Mexican, extracts of Ammi visnaga, ofSiegesbeckia, in particular Siegesbeckia orientalis, vegetable extractsof the family of Ericaceae, in particular bilberry extracts (Vacciniumangustifollium) or Arctostaphylos uva ursi, aloe vera, plant containingsterols (e.g., phytosterol), Manjistha (extracted from plants of thegenus Rubia, particularly Rubia Cordifolia), and Guggal (extracted fromplants of the genus Commiphora, particularly Commiphora Mukul), kolaextract, chamomile, red clover extract, Piper methysticum extract (KavaKava□ from SEDERMA), Bacopa monieri extract (Bacocalmine□ from SEDERMA)and sea whip extract, extracts of Glycyrrhiza glabra, of mulberry, ofmelaleuca (tea tree), of Larrea divaricata, of Rabdosia rubescens, ofEuglena gracilis, of Fibraurea recisa Hirudinea, of Chaparral Sorghum,of sun flower extract, of Enantia chlorantha, of Mitracarpe ofSpermacocea genus, of Buchu barosma, of Lawsonia inermis L., ofAdiantium Capillus-Veneris L., of Chelidonium majus, of Luffacylindrica, of Japanese Mandarin (Citrus reticulata Blanco var. unshiu),of Camelia sinensis, of Imperata cylindrica, of Glaucium Flavum, ofCupressus Sempervirens, of Polygonatum multiflorum, of loveyly hemsleya,of Sambucus Nigra, of Phaseolus lunatus, of Centaurium, of MacrocystisPyrifera, of Turnera Diffusa, of Anemarrhena asphodeloides, of Portulacapilosa, of Humulus lupulus, of Coffea Arabica or of Ilex Paraguariensis.

The compositions of the present invention may include peptides,including, without limitation, the di-, tri-, tetra-, penta-andhexapeptides and their derivatives. According to a particularembodiment, the concentration of the additional peptide, in thecomposition, ranges from 1×10⁻⁷% and 20%, preferably from 1×10⁻⁶% and10%, preferably between 1×10⁻⁵% and 5% by weight. According to thepresent invention, the term “peptide” refers to peptides containing 10amino acids or less, their derivatives, isomers and complexes with otherspecies such as a metal ion (eg copper, zinc, manganese, magnesium, andothers). The term “peptides” refers to both natural peptides andsynthetic peptides. It also refers to compositions that contain peptideswhich are found in nature, and/or are commercially available.

Suitable dipeptides for use herein include but are not limited tocarnosine (beta-AH), YR, VW, NF, DF, KT, KC, CK, KP, KK or TT. Suitabletripeptides for use herein include, but are not limited to RKR, HGG,GHK, GKH, GGH, GHG, KFK, GKH, KPK, KMOK, KMO2K or KAvaK. Suitabletetrapeptides for use herein include but are not limited to RSRK, GQPRor KTFK. Suitable pentapeptides include, but are not limited to KTTKS.Suitable hexapeptides include but are not limited to GKTTKS, VGVAPG.

Other suitable peptides for use herein include, but are not limited tolipophilic derivatives of peptides, preferably palmitoyl derivatives,and metal complexes as aforementioned (e.g. copper complex of thetripeptide HGG). Preferred dipeptide derivatives includeN-Palmitoyl-beta-Ala-His, N-Acetyl-Tyr-Arg-hexadecylester(Calmosensine□, Idealift™, Sederma). Preferred tripeptide derivativesinclude N-Palmitoyl-Gly-Lys-His, (Pal-GKH, Sederma), the copperderivative of HGG (Lamin□, Sigma), Lipospondin (N-Elaidoyl-KFK) and itsanalogs of conservative substitution, N-Acetyl-RKR-NH₂ (Peptide CK+),N-Biot-GHK (Sederma), Pal-KMO₂K (Sederma) and derivatives thereof.Suitable tetrapeptide derivatives for use according to the presentinvention include, but are not limited to, N-palmitoyl-GQPR (Sederma),suitable pentapeptide derivatives for use herein include, but are notlimited to, N-Palmitoyl-KTTKS (MATRIXYL□, Sederma),N-Palmitoyl-Tyr-Gly-Gly-Phe-X with X Met or Leu or mixtures thereof.Suitable hexapeptide derivatives for use herein include, but are notlimited to, N-Palmitoyl-VGVAPG and derivatives thereof. The mixture ofPal-GHK and Pal-GQPR (Matrixyl□ 3000, Sederma) can also be mentioned.

The preferred compositions commercially available containing atripeptide or a derivative include Biopeptide-CL□, Maxilip□, Biobustyl□of Sederma. The compositions commercially available preferred sources oftetrapeptides include RIGIN□, Eyeliss□, Matrixyl□ Reloaded and Matrixyl3000□, which contain between 50 and 500 ppm of Palmitoyl-GQPR andcarrier, proposed by Sederma.

The following marketed peptides can be mentioned as well as additionalactive ingredients: Vialox□, Syn-ake□ or Syn-Coll□ (Pentapharm),Hydroxyprolisilane CN□ (Exsymol), Argireline□, Leuphasyl□, Aldenine□,Trylgen□, Eyeseryl□, Serilesine□ or Decorinyl□ (Lipotec), Collaxyl□ orQuintescine□ (Vincience), BONT-L-Peptide□ (lnfinitec Activos), Cytokino□LS (Laboratoires Serobiologiques/Cognis), Kollaren□, IP2000□ orMeliprene□ (lnstitut Européen de Biologie Cellulaire), Neutrazen□(Innovations), ECM-Protect□ (Atrium Innovations), Timp-Peptide□ or ECMModuline□ (lnfinitec Activos).

Cosmetic Treatment Method

The present invention proposes also a cosmetic treatment method toimprove the general condition of the skin, comprising the topicalapplication to the skin and/or the appendages of an effective amount ofa composition according to the invention as recited above.

The composition of the invention may be applied locally onto areas ofthe face, lips, neck, neckline, hands, feet, or body. One of the majoradvantages of the present invention resides in the ability whenevernecessary or desirable to be able to apply local selective□gentle□treatments through this topical, non-invasive method ofapplication. In the case of anti-wrinkle use for example it may beapplied very locally using a syringe or micro-canula.

It is also possible, however, to consider a composition containing theextract according to the invention intended to be injectedsubcutaneously.

According to other specific features the cosmetic treatment methodaccording to the invention can be combined with one or more othertreatment methods targeting the skin such as lumino-therapy, heat oraromatherapy treatments.

According to the invention, devices with several compartments or kitsmay be proposed to apply the method described above which may includefor example and non-restrictively, a first compartment containing acomposition containing the extract of the invention, and in a secondcompartment a composition containing another active ingredient and/orexcipient, the compositions contained in the said first and secondcompartments in this case being considered to be a combinationcomposition for simultaneous, separate or stepwise use in time,particularly in one of the treatment methods recited above.

For a cosmeceutical or pharmaceutical application, the compositionaccording to the invention is in a suitable form that can be ingested.

The treatment method of the invention is particularly suited to preventand/or treat skin aging by stimulating the detoxification reactionsand/or cell regeneration through a hormetic type response, in particularfor improving the radiance and/or transparency of the complexion, forpreventing and/or treating sensitive and/or reactive skins, forpreventing and/or treating rednesses and/or preventing glycation ofproteins.

A) Example of Obtaining an Extract of Plant Origin According to theInvention by Undifferentiated Cell Culture of Globularia cordifolia

1) Creation of a Cell Line

Globularia cordifolia plants were obtained from seeds.

Leaves are collected, and thereafter cleaned and rinsed with water, anddecontaminated and further rinsed.

a) Step of Induction of Undifferentiated Cells or Callus

Pieces of leaves are cut and placed on the surface of a nutrient agar,containing an assimilable carbon source, a solution of micro andmacro-elements and a combination of adapted hormones and vitamins. Thisnutrient medium, thanks to its composition, will trigger the productionof clusters of undifferentiated cells called callus at the site ofhealing of the leaf.

Composition of the solid nutrient medium used (according to Gamborg):solution of macro-elements and micro-elements, saccharose, agar plant,plant hormones and vitamins.

pH is Adjusted Between 5,5 and 6.

This medium will be thereafter referred to the “basic medium”.

This induction step lasts 1 to 4 months. Culture is made at 25° C. inthe dark.

b) Stabilization and Selection Step of the Cultures in Agar Medium.

Callus stabilization is obtained after successive transplantations,every 3 weeks, on fresh medium. Stable and homogeneous cultures are thusobtained thanks to their phenotypic characteristics (color, friability,proliferation).

The selection is to choose the best callus cultures for the transfer inliquid medium, among the tens initiated earlier. The selected lines arecharacterized by high growth, a soft and crumbly texture, uniform color,good dispersion in liquid medium and stability of these parametersduring the subcultures.

This step lasts from 6 months to 1 year.

c) Transfer in Liquid Medium and Optimization of the Production ofSecondary Metabolites

The selected lines are transferred in liquid medium in order to optimizein an initial stage their growth parameters.

Cell growth and appreciation of the different phases (latence,exponential, stationary) are assessed by regular measurement of thepercentage volume occupied by the culture cells or “packed cell volume”(PCV).

Different suspensions from the callus are placed on an orbital shaker,agitated at 110 rpm in the dark at 25° C. (in Erlenmeyer flasks eachcontaining 100 ml of medium and 10 grams homogeneous texture, color andgrowth callus). Several media and hormone combinations are tested andcompared in order to optimize the growth curve. The optimization of theinitial density of the inoculum is also seeked for reducing the lagphase and thus the global duration of culture.

At the end, the medium chosen is the base medium for having a liquidmedium. Furthermore, the selected initial density for the inoculumcorresponds to a PCV of 10%.

The next step is to determine the optimal environmental conditions todirect the cellular metabolism toward the production of secondarymetabolites at the end of the growth phase of the culture. Thesesecondary metabolites and the other components of the extract medium aswell, will participate in the activity of the extract obtained at theend.

Thus, in the medium, a high proportion of polar molecules wereidentified, including sugars, amino acids, primary metabolites, cinnamicacid and caffeic derivatives, and mixture of phenylethanoid glycosides.The latter were chosen as indicators of secondary metabolites biomassproduction, their total dosage being easily achieved by the method ofArnow, a spectrophotometric method based on the oxidation of totalpolyphenols in alkaline medium by sodium nitrite and sodium molybdate(reading at 510 nm). Other classes of molecules are assumed to bepresent in the medium, such as flavonoids or iridoids but could not beidentified. It is obvious that all of these molecules will beresponsible for the activity of the final extract.

The elicitation chosen according to the invention to lead to theproduction of secondary metabolites is then to place the cells in mediumdepleted of sucrose and macronutrients, preferably in the sameproportions (with a ratio of 1:1).

This transfer phase in liquid medium and optimization of the parametersof growth and production can last from 6 months to 1 year.

After this step, a line giving the best results was selected.

It was subjected to a deposit at DSMZ under the identification referenceof the applicant E68.

2) Extract Obtaining

a) Production of Pre-Cultures in Bioreactors

Successive pre-cultures of the cell line selected of 1-liter, then 5liter, and then 25 liter and finally 100 liters are realized. Each isseeded at 10% PCV and then grown to 50% of PCV. The cell biomass thusproduced is used to inoculate the larger size culture. This stage canlast from 1 to 3 months.

b) Transfer to Bioreactor Comprising the Elicitation Phase

A single use bag bioreactor of 600 liters (nominal volume) is thenseeded between 5 and 20% PVC. The bioreactor is equipped with:

-   A rocking system (rocker) for cell agitation and oxygenation, and    for culture temperature regulation, preferably selected because    being more suitable with regard to the fragility of plant cells    compare to shearing of a conventional bioreactor;-   A control tower equipped with different sensors (oxygen, pH and    temperature) for the follow-up and the regulation of the culture    steps;-   A single-use sterile bag for the cell culture, equipped with    different sensors (pH, oxygen), a filter for gas supply and exchange    systems for sampling or inoculating sterile fluids.

After 10-15 days of culture in the bioreactor, the culture reaches a PCVof about 30 to 45%. The deficient medium is then added to trigger theproduction of secondary metabolites, including here phenylethanoidglycosides. The addition of fresh medium is 10 to 50% of the finalculture volume. After 3-5 days, the culture was stopped at 35-50% ofPCV, the phenylethanoid glycosides then being around 2 grams per literin the culture.

This entire phase in bioreactor lasts 2 to 3 weeks.

c) Recovery of the Extract

The aim of this subsequent step is to produce an aqueous and clarifiedextract.

A cell lysis is achieved, leading to the release of secondarymetabolites in the liquid phase. This lysis is carried out byacidification. A cascade filtration is then carried out, the firstin-depth, followed by sterile filtration, to obtain the aqueous extractof the invention. This step is intended to stabilize the extract.

The extract obtained this way by plant cell culture is clear, colorlessto pale yellow, to the contrary of the “classic” plant extract which isbright yellow, which is an advantage for a direct subsequent formulationof the extract in a cosmetic ingredient or other.

It is Obvious That Variants of This Process Exist, Including:

1) For the extraction of the molecules of interest:

-   The cell lysis step can be performed by biomass thermal treatment,    for example by injecting steam into the biomass. It can also be made    by grinding the cells with microbeads.-   After the cell lysis step, it is possible to purify the crude    extract, in order to enrich it in molecules of interest. According    to this, biomass is first removed by filtration or centrifugation.    The resulting clarified liquid phase is then contacted with an    adsorbent resin, selected for its affinity with the molecules of    interest. This resin is then loaded into a glass chromatography    column. The resin is eluted with appropriate mixtures and the    elution of the product concentrated.-   The recovery of the extract according to the invention can be    implemented using liposomes.

2) Regarding the elicitation mode of the molecules of interest:

The elicitation of compounds of interest can also be done by addingmicrobial fractions to the culture, by adding molecules of biologicalorigin such as, chitosan, methyljasmonate, jasmonic acid, salicylicacid, by adding molecules of non-biological origin such aspaclobutrazol, by applying to the culture a temperature variation, a pHvariation, and/or an osmotic stress induced by a non-metabolizablesugar, such as mannitol, by using an even more drastic impoverishment ofthe environment in macro-elements and sugar, by adding to the cultureadsorbent resins that in addition to elicitation of the compounds ofinterest production can trap them.

3) Instead of the cell line creation stage, a cell line according to theinvention already prepared and preserved can be used. In this case, thestep of creating the cell line is replaced by a conventional step ofamplification of undifferentiated plant cells from said cell lineexisting first in agar and in liquid medium.

The invention method could also be achieved with the other species ofGlobularia.

B) Example of Formulation of a Composition for Implementing theInvention Forming an “Active Ingredient”, a Raw Material for theCosmetics Industry

The active ingredient is a cosmetic composition containing the extractof plant origin of Globularia cordifolia according to the inventiondissolved in a matrix forming a physiologically acceptable medium. Thisactive ingredient is intended for the cosmetics industry for thepreparation of cosmetics, such as creams, gels, etc. (see galenicexamples given below).

The aqueous extract obtained in Example A) above may preferably be mixedat 20 to 80% with any hydrophilic matrix as a gel, an aqueous buffer,glycerin or other short-chain polyol physiologically acceptable.

For example, and for the achievement of the studies and galenicpresented thereafter, an extract up to 33% was preferentially used.

Of course, a pure extract without excipient can also be used as theactive ingredient.

C) In Vitro and Ex-Vivo Studies

The Globularia extract according to the invention has a number ofremarkable effects presented below.

An aqueous extract prepared according to Example B) above, up to 33%,was tested in vitro and showed various cosmetic activities, concerningseveral areas of the skin physiology. The tests were all performed with2% of such Globularia cordifolia extract.

Comparative Tests

First, the superiority of the extract obtained by cell culture versus ahydroalcoholic extract of the whole plant has been shown. These firsttests were carried out by comparing the Globularia cordifolia extractprepared according to B) above (33% of the extract of the invention)diluted to 2%, compared to the hydroalcoholic extract of whole plant atan equivalent concentration in phenylethanoid glycosides.

1) Non-Enzymatic Glycation of BSA

This simple test reproduces in vitro a stress to which regular skinproteins are subjected: their functional alteration by a non-enzymaticglycation phenomenon.

The study model used is a bovine serum albumin (BSA), which is contactedwith a physiological reducing sugar, the fructose, giving a spontaneousand slow reaction between the two molecules that can be accelerated bytemperature.

In this test, the BSA and fructose are incubated in the presence orabsence of the test products at 50° C. for 7 days. The glycation endproducts have a natural fluorescence that can be quantified using afluorimeter (λcx=360 nm and λcm=460 nm).

0.03% Aminoguanidine is used as a positive control of glycationinhibition.

Data are given in Table 1 below.

TABLE 1 Glycation inhibition % Change vs. control Plant extract +8%(nsd*) Globularia cordifolia extract −64% (p < 0.01) *dns =non-significant data

The positive control has a glycation inhibition of −85% versus thecontrol.

2) DPPH Anti-Radical Test

The DPPH (1, 1-diphenyl-2-picryl hydrazyl) is a stable free radicalwhich is widely used for the detection of free radical scavengers. Thismolecule by losing its radical character is converted to DPPH2 (1,1-diphenyl-2-picryl hydrazine). This conversion is accompanied bydiscoloration (purple→yellow) which can be monitored over time byspectrophotometry at 517 nm. Control with no antiradical activity keepsa constant DO. 0003% of caffeic acid is used as a positive control foranti-radical activity. Data are given in Table 2 thereafter.

TABLE 2 Anti-radical activity % Change vs. control Plant extract −1%(nsd) Globularia cordifolia extract +53% (p < 0.01)

The positive control has an anti-radical activity of −85% versus thecontrol.

3) Anti-Singlet-Oxygen Test

Molecular oxygen (O₂) plays a vital role as an acceptor of therespiratory chain terminal electrons. However, this metabolism andothers, as well as external factors, such as UV radiations, lead to theoccurrence of certain derivatives particularly reactive (singlet oxygen,hydroxyl radical, superoxide anion), which will cause oxidative damageto lipids, proteins and nucleic acids, even in healthy bodies. Singletoxygen is one of these very energetic and highly reactive forms ofoxygen. It is possible to produce experimentally in vitro singlet oxygenwith dyes and UVA photons or visible light. The test presentedthereafter uses the Rose Bengal, a dye and visible light.

The purpose of this test is to evaluate the degradation of uric acid bysinglet oxygen formed from the Rose Bengal, under the action of visiblelight. The visualization of the production of singlet oxygen is obtainedby following the destruction of uric acid at 292 nm. In the presence ofa compound capable of neutralizing singlet oxygen, this destruction willbe slowed. The caffeic acid at 0002% is used as a positive control of ananti-singlet-oxygen activity

Data in Table 3 below.

TABLE 3 Anti-singlet-oxygen activity % Change vs. control Plant extract+14% (nsd) Globularia cordifolia extract +33% (p < 0.01)

The positive control has an anti-singlet-oxygen activity of +73% versusthe control.

These three tests show the cosmetic interest of the Globularia extractobtained by cell culture, which also has a much higher efficiencycompared to the hydroalcoholic extract obtained from the plant.

Studies on the Extract of Plant Origin of the Invention

Protection of Keratinocytes in Culture by the Extract of the InventionFacing a Double Stress, Mechanical and Oxidative

Confluent keratinocytes layers were put in contact (test) or not(control) with 2% of the extract of the invention to in a culture mediumfor 24 hours. The layers were then “wounded” in a reproducible mannerand after immediately exposed to UVB to assess their ability to resistto two stresses commonly suffered by the skin. The recovery of celllayers was then evaluated by image analysis, by quantifying the surfacefree of cells (results in Table 4 below).

TABLE 4 Scar surface Wound surface (in arbitrary units) % recolonized T0T48 hours at T48 hours Kerati- Control 43.3 +/− 7.1 19.8 +/− 11.3 54.3%nocytes; Globularia 40.4 +/− 9  2.9 +/− 6.6 92.8% n = 9 cordifoliaextract

The visual observations showed that the control layers underwent aprofound alteration of their integrity (with some cell death) and had agreat difficulty to recolonize the injured surface. To the contrary, thelayers that were in contact with the Globularia cordifolia plant cellextract were less altered and re-form faster a dense and uniform layer.

This preliminary test illustrates well the hormetic phenomenon at workin the presence of the extract according to the invention. Presumably,the cells pre-contact with the extract resulted in the establishment ofa number of lines of defense, including antioxidant, before stress, thenused by the cells to counteract the deleterious effects of stress andregenerate a normal cell layer.

This hormetic effect of the Globularia cordifolia extract has been invitro demonstrated and quantified by studying the following threedistinct axes (4-6).

4-Preservation of Epidermal Progenitors

Principle

The epidermis regenerates continuously in order to produce an effectiveskin barrier. This effect declines with age. The cells at the start ofthis process are called stem cells or progenitor cells. They are rarebut keep their potential to regenerate because of the quality of theirmolecular links binding them to their immediate environment.

Harmful treatments such as oxidants alter the environment of these cellsand make them evolve into a different phenotype, causing a change intheir subsequent ability to proliferate. In vitro these cells, havinglost their proliferative capacity, form small colonies, which may beinvisible to the naked eye, the daughter cells in these colonies beingrelatively large. To the contrary, progenitor cells can multiplyextensively in order to regenerate the skin and produce larger colonieswith smaller cells. This potential for regeneration and thereforeprogenitor cell density in a culture is measured by the cloning efficacymethod. This method involves counting the number of colonies visible tothe naked eye after seeding cells at low density and culturing for aspecific time. The number obtained is used as a measure of theprogenitor vigor of the initial cell population.

Confluent human epidermal progenitor keratinocytes were placed incontact with an optimum medium to force their conversion intodifferentiated keratinocytes (so to make them lose their progenitorphenotype) in the presence or absence of the Globularia cordifoliaextract. Following the contact, the cells of the two series werereseeded at a concentration of 1000 cells/25cm2 (n=5) into thedifferentiating medium.

Results

After culturing for 12 days, the number and size of the colonies can bemeasured after fixation and staining. The results show that the controlcases presented 5.4±1.5 colonies/1000 seeded cells and that the colonieswere small in size. In parallel, contact with the Globularia cordifoliaextract gave 2.3 times more colonies (12.6 colonies±4.3 colonies/1000seeded cells; +133%; p<0.01) than the control and the colonies werelarger in size.

This shows that the Globularia cordifolia extract prevents progenitorcells from converting to a differentiated stage and maintains theirproliferative capacity. It preserves according to this the epidermalcapacity to regenerate itself

5-Stimulation of the Energetic Potential

a. Sirtuin 1

Hormetic effects such as calorie restriction or contact with hormeticmolecules increase sirtuin enzymes and the lifespan of differentorganisms. The sirtuins act particularly on FOXO factor deacetylationand prevent triggering of the production of apoptotic proteins. Thesirtuins also stimulate cellular anti-oxidant defenses and purificationmechanisms (detoxification) and help to preserve mitochondrial functionand therefore energy production. This increases survival and resistanceto stresses.

Human keratinocytes were cultured in the presence or absence of theGlobularia cordifolia extract for 6 days and were then grinded and theintracellular levels of sirtuin-1 were measured using an ELISA method.Results were standardized by parallel measurement of total protein(BCA). Results are given below in Table 5.

TABLE 5 Sirtuin-1 (ng/mg of % Change; protein) significance Kerati-Control 2.10 ± 0.58 Reference nocytes; Globularia cordifolia 3.65 ± 0.33+74%; p < 0.01 n = 5 extract

Positive control Trolox at 500 μm: +27%; p<0.05.

In parallel, abdominal skin explants (58years female) received a topicalapplication daily for 6 days of a cream containing the Globulariacordifolia extract of the invention or its placebo (ex vivo test). Afterthis treatment, the explants were prepared and labeled with fluorescentantibodies to quantify sirtuin-1. This quantification uses an imageanalysis system after photography (50 photos/per case). The results aregiven in Table 6 thereafter.

TABLE 6 Sirtuin-1 (Intensity of % Change; fluorescence). significanceSkin Placebo 10.94 ± 2.40 Reference explants; Globularia cordifolia12.23 ± 2.80 +12%; p < 0.03 n = 5 extract

b. Creatine Kinase

Creatine kinase is a ubiquitous enzyme which exists in two isoforms inthe skin. It is used to store energy in cells and enables tissues torespond to urgent energy requirements. This enzyme converts creatineinto phosphocreatine using ATP which is converted back to ADP. Whennecessary, the enzyme works in the reverse direction and returns energy.Its activity falls with age as it is one of the preferred targets ofreactive oxygen groups Maintaining and increasing the activity of thisenzyme is therefore desirable.

Human dermal fibroblasts were placed in contact with the Globulariacordifolia extract according to the invention for 3 days. The cells werethen exposed without said extract to an oxidative stress (hydrogenperoxide). After a further 3 days □contact with the Globulariacordifolia extract, the cells were again exposed to the same oxidativestress before being homogenized to measure residual intracellularcreatine kinase activity. Activity was measured from the production ofATP from phosphocreatine and ADP. ATP is then measured from an enzymaticreaction leading to the formation of NADPH, readed at 340 nm. Resultswere standardized by measuring total protein (BCA)(results are givenbelow in Table 7).

TABLE 7 Creatin kinase activity (U/mg protein) % Change; significanceFibroblasts; Control without H₂O₂ 0.307 ± 0.020 Reference 1 — n = 5Control with H₂0₂ 0.247 ± 0.030 −19%; p < 0.02 Reference 2 H₂O₂ and0.381 ± 0.055 — +54%; p < 0.01 Globularia cordifolia extract

Positive control Trolox at 500 μm: +28%; p<0.05.

The explants already used for Sirtuin-1 were labeled with fluorescentantibody to assess the amount of mitochondrial epidermal creatinekinase. Quantification was performed using an image analysis systemafter taking photos (50 photos/per case). The results are given in Table8 thereafter

TABLE 8 Mitochondrial creatine kinase (Intensity of % Change;fluorescence). significance Explants; Placebo  7.62 ± 1.70 Reference n =5 Globularia cordifolia 10.81 ± 2.30 +42%; p < 0.01 extract according tothe invention

c. Mitochondrial Membrane Potential

The mitochondrion is a major site involved in the formation of reactiveoxygen groups which, over time, interfere with the functioning of itsrespiratory chain. This causes a decrease in mitochondrial membranepotential (ΔΨ), causing a reduction of the ATP production.

A fall in this membrane potential is a warning sign of ageing and celldeath and it is therefore essential that this be maintained to preservethe mitochondrial ability to produce ATP.

Membrane potential was measured using a specific label sensitive tomitochondrial proton micro-variations. The label exists in two forms,monomeric and polymeric, which have different emission spectra. A highmembrane potential promotes the polymeric form. An increase in themonomer/polymer ratio (520 nm/590 nm) produces therefore a fall in ΔΨ.

A high ratio is therefore less conducive to cell survival than a lowerratio.

The same fibroblast cultures than used for creatine kinase there abovewere used. The results are given below in Table 9.

TABLE 9 Ratio % Change*; significance Fibroblasts; Control without H₂O₂6.15 ± 1.33 Reference 1 — n = 5 Control with H₂O₂ 8.13 ± 1.12 −32%; p <0.01 Reference 2 H₂O₂ and 4.89 ± 0.57 — +40%; p < 0.01 Globulariacordifolia extract

Positive control Trolox at 500 μm: +36%; p<0.05.

(*) % Change=100×[(Control □Globularia cordifolia extract/Control]

Membrane potential therefore improved very markedly with the Globulariacordifolia extract of the invention (+40%, p<0.01).

This study complements the two previous studies to demonstrate theability of the Globularia cordifolia extract of the invention foranti-aging prevention. It stimulates spontaneously the amount ofsirtuin 1. It maintains the activity of creatine kinase and protects theproduction of ATP in response to an oxidative stress.

6-Combatting Noxious Products

a. Endogenous Peroxides

One of the indicators traditionally used to assess the vitality of acell population is the level of oxidative stress. This factor can bemeasured reliably using a probe called DCFH-DA, whose characteristic isto fluoresce in contact with peroxides once it entered the cell.

Protocol

Confluence human keratinocytes were placed in contact or not (negativecontrol) for 24 h with the Globularia cordifolia extract until they grewto confluence. The cells were then exposed to and metabolized the DCFHlabel in a buffer. The label can then react with both intracellularperoxides. The medium was then replaced with one containing or not theGlobularia cordifolia extract and the oxidizing agent model hydrogenperoxide (neither the medium nor the Globularia cordifolia extractreacts directly with H₂O₂). Fluorescence was read in order to estimatethe level of the intracellular peroxide which had reacted with thelabel. The number of cells was quantified using a nuclear labelingmethod (Hoechst 33258). The results are given below in Table 10.

TABLE 10 Intracellular peroxide (UFA/10³ cells) % Change; significanceKerati- Non peroxidized control 37.3 ± 2.2  Reference 1 — nocytes;Negative control with 1099 ± 24.0 X29; p < 0.01 Reference 2 n = 5 H₂O₂H₂O₂ and 137 +/− 4.0 — −88%; p < 0.01 Globularia cordifolia extract

AFU: Arbitrary fluorescence units; Positive control Trolox 500 μm: −91%;(p<0.01).

The Globularia cordifolia extract according to the invention protectsintracellular content from the rise in peroxides induced by hydrogenperoxide. This result reflects a reduction of the damage generated byfree radicals or reactive oxygen species following oxidative stress inthe presence of the Globularia cordifolia extract.

b. SOD and Catalase

Antioxidant enzymes such as catalase or SOD, produced by our bodies, aswell as antioxidants, vitamins E and C, carotenoids, etc. which aredrawn from our food, help to fight against the harmful effects of freeradicals and reactive oxygenated species. So, there is a clear interestin increasing their number by stimulating their synthesis.

A preliminary test on the antioxidant skin potential had shown thathuman keratinocytes treated with the hydrogen peroxide, a model ofoxidative stress, increased their intracellular activity of SOD(superoxide dismutase) by 22%* (p<0.01) compared to the negative controlin the presence of the Globularia cordifolia extract.

*Positive control Trolox at 0.012%; SOD 21%; p <0.01

In addition, explants of abdominal skin (woman, 58 years) received 1topical application daily for 6 days of a cream containing theGlobularia cordifolia extract or its placebo.

At the end of this treatment, the explants were prepared and labeledwith anti-SOD fluorescent antibody on the one hand or anti-catalase onthe other. Quantification was performed using an image analysis systemafter taking photos (50 photos/case). The results are given in Tables 11and 12 below.

TABLE 11 SOD (Intensity of % Change; fluorescence) significance SkinPlacebo 6.31 ± 2.00 Reference explants; Globularia cordifolia 8.58 ±1.40 +36%; p < 0.01 n = 5 extract

TABLE 12 Catalase (Intensity of % Change; fluorescence). significanceSkin explants; Placebo  6.87 ± 2.90 Reference n = 5 Globulariacordifolia 12.24 ± 3.70 +78% p < 0.01 extract

The Globularia cordifolia therefore stimulates SOD and catalaseproduction, independently of any stress. These □spontaneous□stimulation,found in Hormesis, enable the skin to prepare appropriate defensesagainst stresses which may occur later.

c. Proteasome and Glycation

Proteins which are damaged by glycation, oxidation (carbonylatedproteins) or conjugation with lipid peroxides tend to accumulate in thecell during ageing. They are removed routinely by the proteasome whichreduces their harmful effect on cellular homeostasis.The activity of theproteasome, however, is reduced by these accumulations, and byaccumulation of lipofuscin, a lipoprotein waste product, or in time,leading to further increased amounts of damaged proteins by a viciouscycle.

Maintaining the activity of the proteasome is one of the beneficialconsequences of hormesis as it supplements the cells anti-ageing defensearmamentarium. It helps to remove waste which accumulated in the cell,leading to the production of pre-inflammatory substances which opacifythe cells.

Human dermal fibroblasts were placed or not (negative control) incontact with the Globularia cordifolia extract until they grew toconfluence. The Globularia cordifolia extract was then removed and thecells were exposed to the oxidant model, hydrogen peroxide, to produceaccelerated ageing. After this stage, the cells were again exposed tothe Globularia cordifolia extract for 3 days and then were reseeded andsubjected to a further oxidative stress to amplify the effect of theinitial stress.

The cells were then homogenized, and the residual proteasome activitymeasured by monitoring cleavage of a model fluorescent peptide. Resultswere standardized by parallel measurement of protein. Results are giventhereafter in Table 13.

TABLE 13 proteasome activity (UFA/μg proteins) % Change; significanceFibroblasts; Non peroxidized control 1.138 ± 0.15 Reference 1 — n = 5Control with H₂O₂ 0.342 ± 0.04 −70%; p < 0.01 Reference 2 H₂O₂ and 0.538+/− 0.11 — +57%; p < 0.01 Globularia cordifolia extract

AFU: Arbitrary fluorescence units; Positive control Trolox 500 μm: +24%;(p<0.05).

The anti-glycation effect of the extract of Globularia cordifolia wasstudied using a protein model, BSA and a natural reducing sugar,fructose which binds to the protein in a non-enzymatic manner. Theproduct of this binding is fluorescent and is monitored with a recordingsystem. Results are given below in Table 14.

TABLE 14 Glycation (Intensity of % Change; fluorescence) significanceProtein Control 21206 +/− 140 Reference BSA model; Globularia cordifolia 7668 +/− 121 −64%; p < 0.01 n = 4 extract

Positive control aminoguanidine at 0.03%: −85%; p<0.01.

d) Glutathion

In addition to its well-known antioxidant role, glutathion is the mostimportant detoxification element of the body and the principal agent ofthe good health of the body. It binds to toxins such as heavy metals,solvents and pesticides, and transforms them into water-solublecompounds that can easily be eliminated in bile or urine.

Glutathion synthesis was spontaneously stimulated in keratinocytes inthe presence of the cell culture extract of Globularia cordifolia: +49%*(p <0.01) compared to the negative control who did not receive theextract.

*Positive control Trolox at 0.012%: GSH: 53% (p<0.01)

The hormetic type effect (“hormesis-like”) of the extract according tothe invention has thus been shown in particular in 3 directions:

-   A direct strengthening of the molecules and metabolite pathways that    contribute the cells to be more responsive to an ulterior stress    (sirtuins, SOD, catalase, glutathion);-   By direct protection of the energy potential of the cell and its    integrity against oxidative stress (creatine kinase, mitochondrial    potential, proteasome, level of peroxides or glycated proteins) and-   By maintaining the skin regenerative capacity, promoting the    progenitor phenotype and therefore proliferative in cells in charge    of this function.

D) Galenic

Active Ingredient According to the Invention:

Formula comprising the aqueous extract of Globularia cordifoliaaccording to the invention as recited in above example B (at 33%).

Different formulas are described below with or without additionalcosmetic active ingredients, the latter coming as appropriate to supportand/or in addition to the activity of the active ingredient of theinvention. These ingredients can be of any class according to their(s)function(s), site of application (body, face, neck, chest, hands, etc.),the desired final effect and the target consumer, such as anti-aging,anti-wrinkle, moisturizing, circles and/or bags under the eyes, firming,brightening, anti-glycation, anti-spots, slimming, soothing,muscle-relaxant, anti-redness, anti-stretch marks, etc.

EXAMPLE 1 Day Cream Form for Face

% by Ingredients INCI names weight Phase A H₂O Water Qsp100 Ultrez 10Carbomer 0.25 Phase B Butylen glycol Butylene glycol 2.00 PhenoxyethanolPhenoxyethanol qs Phase C Brij S2/Volpo S2 Steareth-2 0.40 BrijS10/Volpo S 10 Steareth-10 1.20 Crodafos CES Cetearyl alcohol & Dicetyl4.00 phosphate & Ceteth 10 phosphate Crodacol CS 90 Cetearyl Alcohol1.00 Laurocapram Azone 2.50 DC 345 Cyclohexasiloxane & 2.00Cyclopentasiloxane Crodamol OSU Ethylhexyl succinate 7.00 Phase DPotassium sorbate Potassium Sorbate 0.10 Phase E H₂O Water 3.00 NaOH 30%Sodium Hydroxide 0.40 Phase F Active ingredient 2.00 comprising theGlobularia cordifolia extract

Procedure: weigh Phase A and let swell without stirring for 30 min. HeatPhase A to 75° C. in a water bath. Weigh and mix Phase B. Weigh Phase Cand heat to 75° C. in a water bath. Add Phase B into Phase A. Mix well.Under stirring add Phase C into Phase (A+B). Homogenize well. Add PhaseD, extemporaneously. Add Phase E, homogenize well. Add Phase F, mixthoroughly.

EXAMPLE 2 Gel Form for the Face

% by Ingredients INCI names weight Phase A H₂O Water Qsp 100 Cetyl Cetyl0.30 hydroxyethylcellulose hydroxyethylcellulose Phase B Ultrez 10Carbomer 0.40 H₂O Water 20.00 Phase C Glycerin Glycerin 3.00 PanstatEthyl & Methyl & Propyl 0.30 parabens Phase D Marcol 82 Mineral oil 4.00Crillet 1 Polysorbate 20 1.00 Crodamol AB C12-15 Alkyl Benzoate 2.00Pemulen TR2 C10-30 Alkyl Acrylate 0.30 cross polymer Phase E Potassiumsorbate Potassium Sorbate 0.10 Phase F H₂O Water 5.00 NaOH 10N SodiumHydroxide 0.50 Phase G Active ingredient 2.00 comprising the extract ofGlobularia cordifolia

Procedure: Disperse Phase A under stirring. Sprinkle Ultrez 10 in water,let stand 30 minutes. Heat Phase C until it is completely dissolved. MixPhase A with Phase B. Add Phase C in Phase (B+A). Add Phase D under inphase (A+B+C). Add Phase E. Neutralize with Phase F. Add Phase G andmix.

Examples of additional ingredients that can be added to the gel typeformula in one of the phases or extemporaneously according to theirhydrophobic or hydrophilic physical property at a certain % depending ontheir concentration and the desired effect:

RIGIN™: active marketed by SEDERMA (WO2000/433417) improving theelasticity and firmness of the skin, increasing hydration and smoothingthe skin. 3% by weight of this ingredient may for example be added tothe formulation in phase G.

RENOVAGE™: global anti-aging active ingredient marketed by SEDERMA(WO2006/020646). 3% by weight of this ingredient may for example beadded to the formulation in Phase D.

LUMISKIN™: active ingredient marketed by SEDERMA (WO2004/024695), whichlightens the complexion. 3% by weight of this ingredient may for examplebe added to the formulation in Phase D.

SUBLISKIN™: active ingredient marketed by SEDERMA (WO2009/055663) thatmoisturizes and smooths the skin while allowing it to resist to externalaggressions. 3% by weight of the ingredient may for example be added tothe formulation in phase G.

MATRIXYL 3000™: anti-wrinkle peptide-based active ingredient marketed bySEDERMA (WO2005/048968) which helps to repair skin damages caused byaging. 3% by weight of this ingredient may for example be added to theformulation in phase G.

EXAMPLE 3 Night Cream Form for Face

% in Ingredients INCI names weight Phase A H₂O Water qsp100 Ultrez 10Carbomer 0.40 Phase B Glycerin Glycerin 3.00 Panstat Ethyl & Methyl &Propyl 0.30 parabens Phase C Polawax GP 200 Cetearyl Alcohol & 1.00polysorbate 20 Crodacol CS 90 Cetearyl Alcohol 1.00 Crodamol STS PPG-3Benzyl Ether 1.00 Myristate DC 200 5 cps Dimethicone 2.50 Crodamol TNIsotridecyl Isononanoate 5.00 Phase D Potassium sorbate Potassiumsorbate 0.10 Phase E NaOH 30% Sodium hydroxide 0.40 H₂O Water 4.00 PhaseF Active ingredient 2.00 comprising the extract of Globularia cordifoliaPhase G Fragrance Fragrance 0.10

Procedure: Weigh Phase A and let swell for 30 minutes. Then heat Phase Ain a water bath at 75° C.; heat Phase B until dissolved. Add Phase B toPhase A. Heat Phase C in a water bath at 75° C. Under stirring, addPhase C in Phase (A+B). Add Phase D, homogenize well. Neutralize withPhase E around 55° C. Add Phase F, then Phase G, homogenize well.

Examples of additional ingredients that can be added to the cream typeformula in one of the phases or extemporaneously according to theirhydrophobic or hydrophilic physical property at a certain % depending ontheir concentration and the desired effect:

DERMAXYL™: anti-aging active ingredient marketed by SEDERMA(WO2004/101609) which smooths wrinkles and repairs the skin barrier. 3%by weight of this ingredient can be added just before use, for examplein phase (A+B+C).

Niacinamide (Vitamin B3), Retinol, resveratrol, DHEA: anti-agingingredients, including anti-wrinkle. 0.5% by weight of retinol,resveratrol or DHEA may for example be added extemporaneously to thephase (A+B+C). 10 wt % of Niacinamide 10% in water for example, can beadded to the phase G.

Hexamidine: anti-bacterial active that can be added to the G phase ofthe formulation to 0.5% by mass.

CHRONODYN™: active ingredient marketed by SEDERMA (WO2006/075311) whichtones and firms the skin, erases visible signs of fatigue. 3% of thisingredient may for example be added to the phase G.

VENUCEANE™: active ingredient marketed by SEDERMA (WO2002/066668) thatprevents visible signs of photo-ageing (spots, wrinkles, dryness, etc.),protects cell structures from damages caused by UV and strengthens skinintegrity. 3% of this ingredient may for example be added to the phaseG.

EXAMPLE 4 Cream Form for the Body

% by Ingredients INCI names weight Phase A H₂O Water qsp 100 Ultrez 10Carbomer 0.40 Phase B Glycerin Glycerin 3.00 Panstat Ethyl & Methyl &Propyl 0.30 parabens Phase C Crill 3 Sorbitan Stearate 2.00 Marcol 82Mineral oil 4.00 Cromollient DP3A PPG 3 Myristyl Ether 1.00 AdipateCithrol GMS AS Glyceryl stearate & 3.00 PEG 100 stearate Phase DPotassium sorbate Sorbate de potassium 0.10 Phase E NaOH 30% Sodiumhydroxide 0.40 H₂O Water 4.00 Phase F Active ingredient 2.00 comprisingthe extract of Globularia cordifolia Phase G Fragrance Fragrance 0.10

Procedure: Weigh Phase A and let swell for 30 minutes. Heat Phase A in awater bath at 75° C.; heat Phase B until dissolved. Add Phase B to PhaseA. Heat Phase C in a water bath at 75° C. Under stirring, add Phase C inPhase (A+B). Add Phase D, homogenize well. Neutralize with Phase Earound 55° C. Add Phase F, then Phase G, homogenize well.

Examples of additional ingredients that can be added to the cream typeformula in one of the phases or extemporaneously according to theirhydrophobic or hydrophilic physical property at a certain % depending ontheir concentration and the desired effect:

JUVINITY™: active marketed by SEDERMA reducing signs of aging on theface and neckline, smoothing wrinkles, densifying and restructuring thedermis. 2% of this ingredient may for example be added extemporaneouslyto Phase (A+B+C).

Tocopherol or vitamin E: actives having anti-radical and antioxidantproperties.

O.D.A. white™: active marketed by SEDERMA (WO1994/07837) which lightensthe skin by reducing the synthesis of melanin. 1% of that ingredient mayfor example be added extemporaneously to Phase (A+B+C).

Tocopherol (vitamin E) or α-lipoic acid (ALA): active with anti-oxidantand anti-radical properties.

0.5% by weight can be added for example to Phase (A+B+C).

Bio-Bustyl™: active marketed by SEDERMA based on peptide and a bacterialfiltrate having a global action on firmness and tone of the bust. 3% ofthis ingredient can be added for example to Phase G.

EXAMPLE 5 Serum Form

% by Ingredients INCI names weight Phase A Optasens G 40 Carbomer 0.25H₂O Water Qsp100 Phase B Butylene Glycol Butylene Glycol 3.00Phenoxyethanol Phenoxyethanol 0.20 Phase C Crillet 1 Polysorbate 20 0.50DC 245 Cyclopentasiloxane 1.00 Crodamol CAP Cetearyl Ethylhexanoate 2.00Crodamol STS PPG-3 Benzyl Ether 0.50 Myristate Pemulen TR2 Acrylates/C10-30 0.20 Alkyl Acrylates cross- polymer Phase D Potassium sorbatePotassium Sorbate 0.10 Phase E H₂O Water 4.00 NaOH 30% Sodium Hydroxide0.45 Phase F Active ingredient 2.00 comprising the extract of Globulariacordifolia Phase G Fragrance Fragrance 0.10

Procedure: Phase A: Sprinkle carbomer in water, let swell 15 minutes.Mix Phase B. Pour Phase B in Phase A and homogenize. Weigh Phase C, mixand add in Phase (A+B), under stirring. Let swell 1 hour.Extemporaneously add Phase D in the previous phase under stirring.Neutralize with Phase E. Put under stirring. Then add Phase F. Allowmixing at least one hour under stirring and then add Phase G. Mix well.

Examples of additional ingredients that can be added to the serum typeformula in one of the phases or extemporaneously according to theirhydrophobic or hydrophilic physical property at a certain % depending ontheir concentration and the desired effect:

LUMISHERE□: active ingredient marketed by SEDERMA (WO04/024695). It isthe combination of diacetylboldine (DAB) encapsulated inpolymethylmethacrylate microcapsules and titanium dioxide modified withmanganese (TiO₂Mn). The TiO₂Mn gives the skin a unifying, mattifying andluminous effect and DAB provides a physiological Phase lighteningeffect. 4% of this ingredient may for example be added to the Phase F ofthe formulation.

REVIDRAT™: active marketed by SEDERMA that in particular improves thecohesion of the epidermis and its hydration. 2% of this ingredient mayfor example be added to Phase C of the formulation.

EVERMAT™: active marketed by SEDERMA (WO2007/029187), which decreasesthe secretion of sebum and thus participates in the treatment of oilyskin. 4% of this ingredient may for example be added to Phase F of theformulation.

HALOXYL™: active marketed by SEDERMA (WO2005/102266), which improves theeye contour by reducing dark circles. 3% of this ingredient may forexample be added to Phase F of the formulation.

EXAMPLE 6 Lotion Form

% by Ingredients INCI names weight Phase A H₂O Water Qsp100 Phase BButylene Glycol Butylene Glycol 5.00 Phenoxyethanol Phenoxyethanol 0.20Phase C Crillet 1 Polysorbate 20 2.00 Crodamol STS PPG-3 Benzyl Ether0.10 Myristate Phase D Potassium sorbate Potassium Sorbate 0.10 Phase EActive ingredient 2.00 comprising the extract of Globularia cordifoliaPhase F Fragrance Fragrance 0.10

Procedure: Weigh Phase A. Weigh Phase B and mix. Add Phase B into PhaseA under stirring for 30 minutes. Weigh Phase C, mix until obtaining ahomogenized mixture. Add Phase C into phase A+B while stirring. AddPhase D in the previous Phase. Add Phase E still under stirring;homogenise well. Weigh Phase F, mix and add to the previous Phase, mixthoroughly.

Examples of additional ingredients that can be added to the lotion typeformula in one of the phases or extemporaneously according to theirhydrophobic or hydrophilic physical property at a certain % depending ontheir concentration and the desired effect:

EYELISS™: an active sold by SEDERMA (WO2003/068141), which helps preventand fight against the appearance of bags under the eyes. 3% of thisingredient may for example be added to Phase E of the formula.

Ac-Net™: an active sold by SEDERMA (WO2003/02828692) offering a completetreatment of oily and acne-prone skins. 3% of this ingredient may forexample be added to Phase E of the formula.

EVERMAT™: mentioned above. 4% of this ingredient can be for exampleadded to Phase E of the formula.

HYDRERGY™: active marketed by SEDERMA (WO2003/02828692 long-termmoisturizer and stimulates the ATP synthesis. 3% of this ingredient mayfor example be added to Phase E of the formula.

E) In Vivo Studies

The tests were achieved with the cream of above example 1.

Principle

The efficacy study of the cosmetic cream containing the activeingredient according to the invention was performed on a panel of 20volunteers with dull skin to assess the improvement in facialcomplexion; on another panel of 22 volunteers with reactive skin todemonstrate an improvement in this effect and on a panel of 14volunteers with visible signs of ageing (wrinkles, spots, etc.) toassess the amounts of SOD and carbonylated proteins present.

Several complementary methods were combined during this study:

-   -   Analysis of clarity and redness of the complexion on        standardised photographs.    -   Analysis of skin transparency by the Transluderm.    -   Analysis of skin shine and matting by the Goniolux.    -   Evaluation of reduced over-reactivity of the skin with the        Neurometer.    -   Evaluation of the amounts of SOD and carbonylated proteins on        adhesive tape strips taken from the cheek.

Protocol

Specific Inclusion Criteria for the Different Studies

Women with a dull, tired and uneven complexion were included in thecomplexion study. 39% of these women were smokers, a factor which isvery often associated with a dull complexion.

In the skin reactivity studies, the volunteers were selected on thebasis of a positive response to the stinging test and from aquestionnaire revealing a sufficient sensitivity score. The volunteerswere required to follow a 15-day washout period (using the placebo).

In the ageing signs study, the 14 volunteers were selected if they wereover 45 years old and had multiple signs of ageing: lines, blemishes orredness. As in the previous study, a washout period was required.

For the 3 tests, no hormonal changes were allowed during the 3 monthsbefore the tests and during the tests themselves (no change incontraceptive, hormone replacement or curative therapy).

Subjects had to use only the cosmetic products provided during thestudy.

Types of Study and Duration

The studies were conducted single blind using non-invasive measurementson:

-   -   20 volunteers with dull skin (mean age 34 years old [range 20 to        48 years old]) who were randomized to apply the cream containing        the Globularia cordifolia extract to half of their face and the        placebo cream to the other half of their face.    -   22 volunteers with reactive skin (mean age 44 years old [range        25 to 64 years old]) who were randomized to apply the cream        containing the Globularia cordifolia extract to one forearm and        the placebo to the controlateral arm. The creams for this panel        were slightly richer (2% glycerine added) because of the        subjects □reactive skin.    -   14 volunteers with physical signs of ageing (average age 57.5        years old [range 47 to 71 years old]) were randomized to apply        the cream containing the Globularia cordifolia extract to one        cheek and the placebo cream to the controlateral cheek.

The cream containing the Globularia cordifolia extract or the placebowas applied by massaging into the skin twice daily for 2 months.

The study is summarized in the diagram below.

T0: Complexion measurements; Neurometer measurements; SOD; Carbonylatedproteins

T 1 month: Neurometer measurements

T 2 months: Complexion measurements; SOD; Carbonylated proteins

Statistical tests were performed using the Student t test or with anon-parametric Wilcoxon test where necessary. In both situations, thetests were single-tailed and on paired measurements.

1.1 Study of Facial Complexion

Analysis of Clarity and Redness from Standardised Photographs

A photography system, the HeadScan was used to take identicalphotographs on the two times. The system uses a high definition digitalcamera equipped with flashes and filters to obtain cross-polarized lighttaking a shine-free image.

For each half of the face, 5 areas were automatically extracted with theFrameScan™ software to calculate the Clarity (L*) and Redness (a*)indices.

The results are given in the Tables 15 and 16 below.

TABLE 15 Change in clarity of facial complexion after application for 2months of the cream comprising the extract of Globularia cordifolia; (N= 20) Improvement in clarity of complexion L* T0 T 2 months PLACEBOcream 29.86 ± 2.46 27.36 ± 2.17 % Change (significance) −8.4%; (p <0.01) Globularia cordifolia cream 29.88 ± 3.79 30.12 ± 3.39 % Change(significance) 0.8% (dns)   Difference (Globularia cordifolia 9.2%extract cream − Placebo) of the difference of % variation vs. T0.Significance vs. placebo p < 0.01 *Physiological scale: the first 20units of the scale represent a very dull surface; these are deducted.

The results show that complexion clarity, measured by the parameter L*,is deteriorated on the placebo side after 2 months, with a difference of8.4% from T0 (p<0.01). On the side of the extract of Globulariacordifolia according to the invention, clarity remained stable duringthis period when skin parameters deteriorated. A marked improvement(+9.2% (p<0.01)) in facial clarity was seen against placebo.

TABLE 16 Change in redness of facial complexion after application of thecream comprising the extract of Globularia cordifolia for 2 months; (N =20) Improvement in Redness + a* T0 T 2 months PLACEBO cream 14.42 ± 2.6116.89 ± 1.95 % Change (significance) 17.1% (p < 0.01) Globulariacordifolia 14.28 ± 2.32 14.81 ± 2.07 extract cream % Change(significance) 3.7% (dns)    Delta (Globularia cordifolia −13.4% extractcream □ Placebo) of the difference of % variation vs. T0. Significancevs. placebo p < 0.01

In parallel, redness complexion of these volunteers increases on theplacebo side during the test by +17.1%; (p<0.01). This may have been dueto the different weather conditions between the two measurement times.Application of the Globularia cordifolia extract cream however limitedthis increasing trend in redness. The difference between the two sideswas in favor of the side treated with the Globularia cordifolia extractcream: −13.4%; (p<0.01)

From the results obtained for these two parameters, fewer imperfectionsand redness with a more even and radiant complexion for the face can bededucted.

Analysis of Skin Transparency by TRANSLUDERM□

Skin transparency was assessed by an instrument developed by OrionConcept: the Transluderm□. This instrument consists of a 17 mm radiusplate, the center of which contains a continuous spectrum white lightdiode which illuminates the skin. Sensors are arranged every 1.5 mm inall four directions, recording the amount of light given off by theskin. An image is taken using a high resolution camera and imageanalysis is performed with dedicated software. A Cinaximum propagationdistance□parameter of light conducted through the skin is obtained fromthis.

Understandably, transparent skin conducts light further.

Results are given in table 17 below.

TABLE 17 Change in skin transparency of measured skin by theTransluderm□ after application of the Globularia cordifolia extractcream; (N = 20) Distance (mm) T0 T 2 months PLACEBO cream 18.90 ± 13  21.59 ± 11.4 % Change (Significance) +14.3% (dns)     Globulariacordifolia 19.1 ± 12.4  31.9 ± 31.9 extract cream % Change(significance) +67% (p < 0.03) Delta (Globularia cordifolia 52.7%extract cream □ Placebo) of the difference of % variation vs. T0.Significance vs. placebo p = 0.095

The light was found to travel a far greater distance (+52.7% vs.placebo) indicating that skin transparency is greatly improved afterapplying the Globularia cordifolia extract cream. The previouslyobserved effects are thus reinforced.

Analysis of Skin Radiance by the GONIOLUX™

The Goniolux™ (Orion Concept) was used to assess shine and mattingparameters. This instrument quantifies the interaction between lightprojected onto the skin (incident light) and the behaviour of the lightreturned by the skin (reflected light). Reflected light is measuredusing multiple sensors in all directions in a three-dimensional space.The light reflected can be broken down into specular (mirror effect)reflected light and diffuse reflected light (halo effect). The skinappears more youthful and more beautiful when the light returned is softwith less shine and more matting (halo). Results are given below inTable 18.

TABLE 18 Change in the specular light and diffuse light parameters afterapplying the Globularia cordifolia extract cream; (N = 20) Shine Satineffect (arbitrary unit) (arbitrary unit) T0 T 2 months T0 T 2 monthsPLACEBO cream 780.7 ± 8.2 750.2 ± 27.2 7.37 ± 0.06 8.22 ± 0.51 % Change(Significance) −3.9% (dns)   +11.5% (dns)   Globularia cordifolia 777.0± 8.2 706.0 ± 28.4 7.39 ± 0.06 8.71 ± 0.51 extract cream % Change(significance) −9.1% (p < 0.02) +17.9% (p < 0.01) Delta (Globulariacordifolia +5.2% +6.4% extract cream − Placebo) of the difference of %variation vs. T0. Significance vs. placebo p = 0.07  Trend (p < 0.15)

Application of the Globularia cordifolia extract cream for 2 monthsproduced a beneficial mattifying effect consisting of reduced shine(−9.1%; p<0.02 vs. T0), producing an improvement in results for thisparameter (+5.2%; p<0.07 vs. placebo) combined with an increasing trendin the matt appearance (+17.9%; p<0.01 vs. T0).

Improvement in these 2 parameters produces a face which is smoother andsofter in appearance.

1.2 Study of Skin Reactivity

In order to examine the improvement in volunteers' skin reactivity, aninstrument called the NEUROMETER™ was used, this instrument being usedin medicine for early detection of hyper or hypo-reactivity.

The NEUROMETER™ can be used to provide both a quantitative andqualitative characterization of changes in skin sensitivity.

The instrument stimulates nerve cells by delivering a small current tothe skin until the volunteer detects something (the procedure isentirely painless). This corresponds to the individual currentperception threshold (or CPT=Current Perception Threshold).

If a person can perceive currents at a higher intensity than otherpeople, this indicates that they have a higher skin tolerance tostresses and a less reactive skin. Conversely, low perception currentsindicate a more reactive skin.

There are several types of nerve fibers, (Aβ, aδ and C fibers) which canbe excited by different frequency currents. The 3 types of nerve fiberscarry different information. Because of the experiences of volunteers inthe inclusion tests, forearm type Aβ fibers as the model in this studywas used. These large diameters, rapid conduction fibers, carry skintouch, pressure, vibration and distension information. They areassociated with four types of mechano-receptors: Meissner's corpuscles,Ruffini's corpuscles, Merkel's disks and Pacini's corpuscles.

Measurements were performed automatically and double-blind in thisstudy, ensuring very reliable results, which are particularly importantfor this type of individually dependent, subjective sensation. Resultsare given in below Table 19.

TABLE 19 Change in skin tolerability threshold after applying theGlobularia cordifolia extract cream; (N = 22) Aβ fibers (in mAmperes) T0T 1 months PLACEBO cream 81.82 ± 17.9 75.64 ± 17.9 % Change(Significance) −7.6% (dns)     Globularia cordifolia 69.45 ± 16.8 80.55± 22.9 extract cream % Change (significance) +16% (p < 0.01) Delta(Globularia cordifolia +23.6% extract cream − Placebo) of the differenceof % variation vs. T0. Significance vs. placebo p < 0.01

The results show a slight and insignificant deterioration in theperception threshold on the placebo site which, as a result of thisfall, makes the skin more reactive than at T0.

Conversely, on the site which was treated with the cream comprising theGlobularia cordifolia extract, the perception threshold rose verysignificantly by +23.6%; (p<0.01) compared to placebo. This indicates afall in skin reactivity after using the Globularia cordifolia creamaccording to the invention.

1.3 Studied SOD (Enzyme Superoxide Dismutase) and Carbonylated Proteinson Adhesive Tape Strips

A test was conducted on volunteers with visible signs of ageing (N=14)to confirm the stimulation of SOD production seen in culture cells andin explants treated with the Globularia cordifolia extract cream.

After applications of the cream containing the Globularia cordifoliaextract onto the cheek for 2 months, SOD activity measured from adhesivetape strips taken from the cheeks showed a marked improvement inactivity of +19% on the site of the Globularia cordifolia extract creamcompared to a fall of 13% on the controlateral placebo site. Thedifference of 32% is significant (p<0.01).

Furthermore, the amounts of carbonylated proteins found on the stratumcorneum cells removed by adhesive tape strips in the same study werealso measured using the method of FUJITA et al, (2007). Carbonylatedproteins were identified directly on the adhesive tape strips using anFTZ fluorescent label and measured with a fluorescence reader. Data wasstandardized by parallel measurement of total protein. The results aregiven below in Table 20.

TABLE 20 Change in amounts of carbonylated proteins after applying theGlobularia cordifolia extract cream; (N = 14) Carbonylated proteins/mgprotein (fluorescence units) T0 T 2 months % Change/T0 PLACEBO cream158910 +/− 89197 184364 +/− 93933 +16%  % Change (significance)Globularia cordifolia 170510 +/− 77739 167937 +/− 77098 −2% extractcream % Change (significance) Delta (Globularia cordifolia 18% extractcream − Placebo) of the difference of % variation vs. T0. Significancevs. placebo p < 0.01

These results show that less carbonylated protein was collected onto theadhesive strips on the side which was treated with the Globulariacordifolia extract cream and that this difference was significantcompared to the placebo site (−18% on the placebo side; (p<0.01).

Thus, after 2 months' application to skin with visible signs of ageing,the Globularia cordifolia extract cream is able to improve SOD activityand reduces amounts of carbonylated proteins present. Combined with theobservations in the tests on dull skin, it can be concluded that theGlobularia cordifolia extract cream has stimulated the skin's defencesand restored its transparency and radiance. The improvement in cleansingcapacity has helped to reduce reactiveness, making the skin lessreceptive to future stresses, as being immunized. The Globulariacordifolia extract cream therefore acts as a genuine anti-ageingvaccine.

1-25. (canceled)
 26. A composition comprising: a cellular extract ofundifferentiated cells from culturing callus of a Globularia cordifoliaplant, the cellular extract comprising an enhanced content ofphenylethanoid glycosides as secondary metabolites.
 27. The compositionof claim 26 wherein said composition further comprises a physiologicallyacceptable medium or matrix.
 28. The composition of claim 26 whereinsaid composition is formulated for topical use.
 29. The composition ofclaim 26, wherein the composition comprises a physiologically acceptablesolubilizer selected from the group consisting of: ethanol, propanol,isopropanol, propylene glycol, glycerin, butylene glycol, orpolyethylene glycol, and any combination thereof.
 30. The compostion ofclaim 27, wherein the physiologically acceptable solubilizer isglycerin.
 31. The composition of claim 26, wherein the extract ispresent in the amount of 0.0001% to 10% by weight of the composition.32. The composition of claim 27, wherein the composition comprises ahydrophilic matrix comprising the extract and gylcerin.
 33. Thecomposition of claim 32, wherein the extract is present in the amount of20 to 80% of the hydrophilic matrix.
 34. The composition of claim 32,wherein the extract is present in up to 33% of the hydrophilic matrix.35. The composition of claim 26, wherein the extract is derived fromcell line DSM 25009.